1. INTRODUCTION
Inflammation is a crucial biological reaction that protects the body from harmful agents, whether they originate internally or externally. The process begins with the activation of different cells and the subsequent release of chemical signaling molecules, notably including prostaglandins, leukotrienes, and macrophages. These, in turn, promote the overproduction of proinflammatory cytokines, such as interleukins (IL)-1β, IL-6, and tumor necrosis factor (TNF), as well as inflammatory mediators like reactive oxygen species (ROS) and nitric oxide (NO•). This response is a defensive mechanism intended to eradicate the causative agent. However, when inflammation becomes chronic and exacerbated, it becomes an underlying factor in the development of various chronic noncommunicable diseases (NCDs), such as diabetes, atherosclerosis, asthma, cardiovascular disease, inflammatory bowel disease, rheumatoid arthritis, and even cancer [1–3]. The World Health Organization (WHO) recently estimated that chronic NCDs cause 43 million deaths each year, making up 75% of all deaths worldwide, mostly affecting individuals aged 30–69. According to the WHO, the leading causes of death in this age group are cardiovascular diseases, cancer, chronic respiratory diseases, and diabetes [4]. These pathologies develop gradually, and their clinical symptoms usually appear only after significant tissue damage has already taken place, which greatly impairs patients’ quality of life and can even result in death [3,5].
The pharmaceutical agents used to treat the inflammation are categorized into three main classes: steroids, nonsteroidal anti-inflammatory drugs (NSAIDs), and cytokine receptor antagonists. Although NSAIDs are the primary treatment choice, their use is associated with a high rate of adverse gastrointestinal, cardiovascular, and renal effects [6]. Therefore, there is an ongoing and urgent need to discover new substances and develop innovative formulations that are safer and effective for treating diseases related to chronic inflammation [7]. Plants have been demonstrated to be a valuable source of bioactive compounds, which possess significant therapeutic potential. A significant number of these compounds have already been isolated, identified, and successfully included in the pharmaceutical industry. In the domain of inflammation treatment, flavonoids, the most prevalent class of plant phenols, have demonstrated notable anti-inflammatory properties. Interest in traditional medicine is increasing primarily due to consumer preference for natural alternatives, concerns about the side effects of allopathic medicine, and the lower cost of therapies based on natural products. However, numerous plant extracts exhibit limited chemical and physical stability, a circumstance that has the potential to compromise their therapeutic efficacy, restrict their shelf life, and impede their incorporation into conventional pharmaceutical formulations [8].
Ambrosia peruviana is a perennial, aromatic plant native to Central and South America, used in traditional medicine to treat conditions involving inflammatory processes. Previous studies by our research group showed that the total ethanolic extract of its seeds significantly inhibits the production of important inflammatory mediators such as NO•, TNF-α, IL-1β, IL-6, and prostaglandin E2 in RAW 264.7 macrophages stimulated with lipopolysaccharide (LPS), providing a starting point for the design and development of phytotherapeutic products with anti-inflammatory activity. We have previously demonstrated the presence of alkaloids, flavonoids, tannins, coumarins, cardiotonic glycosides, saponins, triterpenes/steroids, quinones, and polyphenolic compounds in the seeds extract with the latter being particularly associated with anti-inflammatory and antioxidant biological activities. In addition, we conducted a preformulation study that allowed us to select compatible excipients for the development of a topical formulation with anti-inflammatory activity using this extract [9,10].
Considering that microemulsions (MEs) offer several advantages, including increased drug solubility, enhanced thermodynamic stability, longer shelf life, quick onset of pharmacological effects, and improved safety [11]. These characteristics make MEs promising carriers for topical phytotherapeutic products.
Although a few studies have utilized D-optimal mixture designs to develop ME systems with plant extracts, the present work introduces a distinct and innovative contribution. To our knowledge, this is the first ME formulation developed using the seeds extract of A. peruviana, a specie whose chemical profile and pharmacological activity. have not previously been explored in ME systems. Additionally, our formulation integrates excipients selected through a prior evidence-based preformulation study and confirms preservation of anti-inflammatory activity in vitro. These elements establish the novelty of this study and clearly differentiate it from previous reports using botanical extracts in optimized MEs.
Therefore, this study aimed to develop, optimize, and characterize an anti-inflammatory ME containing A. peruviana seeds extract, evaluating droplet size, physicochemical properties, and biological activity.
2. MATERIALS AND METHODS
2.1. Materials
Ethanol and Dimethyl sulfoxide (DMSO) were purchased from Thermo Fisher Scientific, Dulbecco’s modified eagle medium (DMEM), fetal bovine serum (FBS), penicillin, streptomycin, gallic acid, sodium carbonate, LPS from Escherichia coli, 4-aminobenzenesulfonamide, N-[1,1-naphthyl] ethylenediamine dihydrochloride, sodium nitrite, N-(3-(amino methyl) benzyl) acetamidine (1400 W), and Folin Ciocalteu were obtained from Sigma Aldrich. Macrophages RAW 264.7 (TIB-71TM) were acquired from the American Type Culture Collection. Bromide of 3[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium was obtained from Calbiochem®. The excipients including castor and coconut oils were also supplied by Sigma Aldrich, Ceteareth-20 (Eumulgin® B2), PEG-150 distearate (Eumulgin® EO 33), poloxamer 188 (Kolliphor® P188), PEG-40 hydrogenated castor oil (Cremophor® RH-40), cocamide DEA (Comperlan® KD), [lauryl ether sulfate-MIPA, lauryl ether-4 and cocamide DEA] (Plantapon® WW 1000), poloxamer 407 (Kolliphor® P407), [tocopherol and hydrogenated glycerides palm citrate] (Controx® KS C), phenoxyethanol and Ethylenediaminetetraacetic acid (EDTA) were purchased from BASF. Polysorbate 80, glycerin, and propylene glycol were supplied by Dow Chemical Company.
2.2. Plant extraction and phytochemical screening
Ambrosia peruviana seeds were collected in Turbana, Bolívar, Colombia (10°24′0″ N, 75°30′0″ O), in July 2018. A voucher specimen of the plant was identified in the herbarium of the Universidad de Antioquia (Medellín, Colombia) by the biologist Felipe Cardona and deposited with the identification code HUA 214539. The seeds of A. peruviana were extracted repeatedly with 96% ethanol (v/v) through maceration at room temperature (25°C ± 3°C) until exhausted. The resulting extract was then dried using a rotary evaporator (Heidolph, Germany) set at a constant 40°C, 250 mbar in 50 ml round-bottomed flasks, and stored at −20°C. A preliminary phytochemical analysis was conducted to confirm the presence of major chemical groups using standard precipitation and colorimetric methods. The results aligned with those reported previously [9], indicating the presence of various bioactive compounds, including alkaloids, flavonoids, tannins, coumarins, cardiotonic glycosides, saponins, triterpenes/steroids, and quinones (Data not shown).
2.3. Formulation study
The excipients used in this formulation study were chosen based on their prior proven compatibility with A. peruviana extract in a preformulation study conducted by our research group (Table 1) [9].
Table 1. List of excipients compatible with the
extract of A. peruviana All. seeds.
| Excipient | Function |
|---|---|
| Castor oil Coconut oil | Oily phase |
| PEG-150 distearate Poloxamer 188 Hydrogenated castor oil PEG-40 Cocamide DEA Lauryl ether sulfate-MIPA, lauryl ether-4 and cocamide DEA Polysorbate 80 Ceteareth-20 | Surfactant |
| Propylene glycol Poloxamer 407 Glycerin | Co-surfactant |
| Tocopherol and hydrogenated glycerides palm citrate | Antioxidant |
| Benzyl alcohol, methylchloroisothiazolinone and methylisothiazolinone | Preserving agent |
| EDTA | Stabilizing agent |
| Water | Vehicle |
Table adapted from previous results of a preformulation study of A. peruviana seeds [9].
2.3.1. Selection of the oil
The selection of oil in the formulation was based on assessing the solubility of the A. peruviana extract in coconut and castor oils. An excess of the dry extract was added to 1 ml of each oil in separate vials, and the mixtures were thoroughly mixed using a vortex mixer (BOECO V1-Plus). The vials were kept at 25°C ± 1°C in a shaking water bath for 72 hours to reach equilibrium solubility. Afterward, the samples were centrifuged at 3,000 rpm for 15 minutes, and the supernatant was filtered through a 0.45 µm PTFE membrane filter.
The dissolved extract content was quantified using the total polyphenol content method, expressed as gallic acid equivalents. Briefly, an aliquot of the filtered supernatant was appropriately diluted in methanol, and the total polyphenol concentration was determined using the Folin–Ciocalteu colorimetric method at 760 nm. All measurements were performed in triplicate [9,10].
2.3.2. Surfactant selection
The selection of surfactant was based on its ability to incorporate the maximum amount of oily phase in a fixed volume of water without destabilizing the system. To achieve this, 10% w/w solutions of surfactants were prepared using distilled water, and the oil, chosen in the previous step, was added to the surfactant solutions in successive 20 μl increments. After each addition, the mixture was shaken, and its appearance was visually evaluated. The appearance of turbidity was considered the endpoint of the assay.
2.3.3. Co-surfactant selection
Considering the synergistic nature of the co-surfactant with the surfactants in the ME formulation, the selection of this component was also conducted. To this end, a mixture of the selected surfactant and the different co-surfactants was prepared in a weight ratio of 75:25 (S/CoS) in 10% w/w solutions using distilled water. Subsequently, 20 μl of oil was added incrementally. The mixture was then stirred, and the appearance of turbidity or phase separation in the resulting mixture was considered the endpoint of the experiment. All tests were performed in triplicate.
2.4. Microemulsion formulation
2.4.1. Preparation of the pseudoternary phase diagram and selection of components
Following the preliminary selection of the oil, S/CoS, pseudoternary phase diagrams were constructed using the titration method with distilled water as the aqueous phase. To this end, S/CoS mixtures were meticulously prepared in five weight ratios: 1:1, 1:2, 1:3, 2:1, and 3:1. These mixtures were then combined with the selected oil in ratios ranging from 1:0 to 0:1. The system was subjected to a slow titration with water, agitated with a vortex mixer and subjected to a visual inspection after each addition, noting the transition from transparency to turbidity. The resulting spots allowed for the delineation of the regions of ME formation [12,13]. A 3:1 S/CoS ratio was selected for further development because it yielded the most extensive region of ME formation. This finding was used to establish the ratio ranges for a D-optimal mixture design, which aimed to optimize the ME’s critical variables [14]. The pseudoternary phase diagrams were constructed using OriginPro, Version 2025b (64-bit) SR1, Learning Edition (OriginLab Corporation, Northampton, MA; Version 10.2.5.234).
2.4.2. D-optimal mixing design (DOMD)
DOMD is a statistical approach that is used to optimize formulations in which components are mixed in varying proportions [15]. The factors to be evaluated included X1 (oil), X2 (water), and X3 (S/CoS mixture), which were utilized at two levels: high and low. The sum of X1, X2, and X3 was maintained at a constant value of 100% [16]. The following were used as response variables: droplet size, extensibility, and physical stability of the obtained MEs.
2.4.3. Evaluation of response variables of DOMD
2.4.3.1. Droplet size
For the determination of droplet size in A. peruviana, ME was accomplished through the implementation of the dynamic light scattering technique, employing the particle analyzer equipment (Malvern Instruments Inc. Zetasizer Nano ZS) in accordance with the methodology previously outlined. The measurements were conducted at a temperature of 25°C on samples that had been diluted in ultrapure water. This approach was taken to minimize the viscosity of the medium and to mitigate potential interactions between droplets that could compromise the accuracy of the analysis. To ensure the reproducibility of the data, three replicate measurements were performed for each sample. This technique enabled the calculation of the average hydrodynamic droplet size, expressed in nanometers (nm), thereby providing an accurate estimate of the physical behavior of the ME in terms of its globular size [17].
2.4.3.2. Extensibility
Extensibility was measured using two glass plates (8 × 4 cm) and weights of 2, 5, and 10 g. Initially, the lower plate was placed on a millimeter sheet, and the diagonals were traced. Then, 45 mg of MEs were placed over the insertion point. The upper plate was then positioned, and after a 30 seconds interval, the diameter values that had been achieved were recorded. This procedure was repeated with the subsequent weights, with a 30 seconds interval observed between the application of each new weight and the subsequent diameter measurement. Each determination was made in triplicate.
2.4.3.3. Physical stability
The stability of the DOMD formulations was evaluated by observing turbidity or phase separation. For this purpose, four consecutive cycles of heating (45°C) in an oven (Thermo Scientific Heraeus UT20) and cooling (4°C) in a freezer (Thermo Scientific TSX) were performed. Each cycle consisted of 24 hours of processing time. Furthermore, a centrifugation test was conducted at 3,200 rpm for a duration of 30 minutes on days 0, 3, 7, and 14 using the centrifuge (Clay Adams Compact II).
2.5. Preparation of microemulsion
DOMD facilitated the selection of the optimal combination of oil, S/CoS, and water. The oil, the selected S/CoS mixture, and the dry extract of A. peruviana seeds were thoroughly combined to formulate the oil phase using a vortex mixer. The components were mixed for 5 minutes at medium vortex speed under ambient temperature conditions (25°C ± 2 °C). Subsequently, water was gradually incorporated, and the mixture was stirred using the vortex mixer at medium speed for an additional 5 minutes to obtain the final ME formulation [14].
The resulting ME was characterized in terms of droplet size, extensibility, and physical stability. Additionally, cell viability and NO• inhibition assays were performed.
2.6. Formulation characterization
The viscosity of the ME was evaluated using a Brookfield viscometer (Brookfield, Stoughton, MA) at 25°C ± 1°C, employing Spindle No. 4 at approximately 30 rpm. All measurements were performed in triplicate.
The pH of the ME was determined using a calibrated potentiometer (OHAUS Starter 3100, OHAUS Corp.). Measurements were performed at 25°C ± 1°C by immersing a combination glass electrode directly into the sample until a stable reading was obtained. All measurements were carried out in triplicate. Electrical conductivity was measured using a conductivity meter (JSI1006, Jyoti, India) to determine the type of ME. The refractive index was determined using an Abbe refractometer (Scientech, India). All measurements were carried out in triplicate at room temperature (25°C ± 1°C). The physical stability of the formulation was evaluated using three specific tests: (i) heating and cooling cycles (4°C and 45°C, with 4 to 6 cycles of 48 hours at each temperature, in a refrigerator); (ii) centrifugation at 3,200 rpm for 30 minutes; and (iii) freezing and thawing cycles (−21°C and 25°C, with storage of 48 hours in each phase, in a freezer). In each test, the appearance of turbidity or phase separation was observed as an indicator of instability.
2.7. Evaluation of anti-inflammatory activity
The biological evaluation of the ethanolic extract of A. peruviana and its topical formulation was carried out using RAW 264.7 murine macrophages stimulated with LPS, as an in vitro model of inflammation. The cells were cultured in DMEM, a nutrient-rich solution that contains 10% inactivated FBS and 1% penicillin/streptomycin, to ensure optimal cell growth. These conditions remained constant during stages of seeding, pre-incubation, and experimental treatment periods. The cultivation environment was maintained at 37°C in a humidified atmosphere with 5% CO2, as previously described [9].
2.7.1. Cell viability assay (MTT)
The assessment of cell viability was conducted through the implementation of the MTT assay. At the end of treatment and incubation, the medium was replaced with fresh DMEM containing MTT at a final concentration of 0.25 mg/ml. The plates were incubated for 4 hours at 37°C, the medium was removed, and the formazan crystals formed were dissolved in 100 μl of DMSO. The absorbance was measured at 550 nm. Cell viability was expressed as a percentage relative to the untreated control group. The inclusion of a placebo formulation was deemed necessary to evaluate the potential influence of excipients on cell viability and NO• production. All experiments were performed in duplicate (n = 2), and each experimental condition was evaluated with six replicates per trial (n = 12).
2.7.2. NO• inhibition assay
NO• production was indirectly assessed by quantification of accumulated nitrite in the culture medium using the colorimetric Griess reaction [18]. RAW 264.7 cells were seeded in 24-well plates at a density of 2 × 105 cells/well, and then subjected to a 24 hours preincubation period. They were then treated with the formulation, a placebo formulation (excipients without extract), and 1400 W (10 μM) as a positive control for 30 minutes. Subsequently, the cells were stimulated with LPS (1 μg/ml) and incubated for an additional 24 hours. Supernatants were collected and stored at −20°C until analysis. For the assay, equal volumes of the supernatant and Griess reagent (1% sulfanilamide in 5% phosphoric acid and 0.1% N-(1-naphthyl) ethylenediamine dihydrochloride) were mixed in 96-well plates and incubated at room temperature for 10 minutes. The absorbance was measured at 550 nm using a microplate reader (Multiskan GO, Thermo Scientific), and the nitrite concentration was calculated from a NaNO2 standard curve (1–200 µM).
2.8. Statistical treatment
Results are expressed as the mean ± standard error of the mean (SEM) of three (DOMD and pharmaceutics characterization assay) and two (in vitro assay) independent experiments. Data were analyzed by one-way analysis of variance (ANOVA) followed by Dunnett’s post hoc test. In all cases, statistically significant differences were considered to be those with a p-value < 0.05.
3. RESULTS AND DISCUSSION
3.1. Formulation study
3.1.1. Selection of excipients
MEs are quaternary nanocolloidal systems that are thermodynamically stable, consisting of an oily phase and an aqueous phase, containing S/CoS. They have globule sizes ranging from 10 to 200 nm. Every component of ME plays an important role. The solubility of the ethanolic extract of A. peruviana seeds was found to be higher in coconut oil (49 ± 0.2 mg extract/g oil) compared to castor oil (33 ± 0.3 mg/g). For this reason, coconut oil was selected as the oil phase of the formulation. The composition of the oils greatly affected the solubility of the extract. Although both oils contain fatty acids such as oleic, linoleic, palmitic, and stearic acids, coconut oil is notable for having short-chain fatty acids, unlike castor oil, which mostly has long-chain fatty acids. Since the extract is polar, it dissolves better in short-chain fatty acids like those in coconut oil because of the more available hydroxyl groups that can form bonds [19,20].
For surfactants, the choice was limited to nonionic types, taking into account their lower toxicity compared to ionic types, and considering their capacity to incorporate the oil as a key criterion [14,21]. Surfactants such as lauryl ether sulfate-MIPA, lauryl ether-4, and cocamide DEA, along with those that did not dissolve in 10% water, were excluded from further testing. Polysorbate 80 showed the highest oil-incorporation capacity (110.33 ± 2.13 mg/g), followed by poloxamer 188, ceteareth-20, and PEG-40 hydrogenated castor oil (Table 2). Therefore, polysorbate 80 was selected as the surfactant for developing the ME in this study.
Table 2. Oil-incorporation capacity of surfactants.
| Surfactant | Oil-incorporation capacity (mg/g) * |
|---|---|
| Poloxamer 188 | 71.87 ± 0.72 |
| Ceteareth-20 | 64.42 ± 1.34 |
| PEG-40 Hydrogenated castor oil | 1.61 ± 2.39 |
| Polysorbate 80 | 110.33 ± 2.13 |
* Each value represents the mean ± SD of three independent measurements (n = 3).
The capacity of the evaluated surfactants to incorporate coconut oil is related to their chemical structure and hydrophilic-lipophilic balance (HLB). The HLB is indicative of the surfactant’s affinity for both the oil and aqueous phases; a higher HLB value corresponds to a greater degree of hydrophilicity. In this instance, polysorbate 80, which exhibited a lower HLB compared to the other surfactants under consideration, demonstrated a higher oil-incorporation capacity. This finding indicates a potentially more robust interaction with the oily phase [22,23].
The selection of the co-surfactant was based on its ability to reduce interfacial tension in combination with polysorbate 80, as well as its physical form, prioritizing liquids over solids due to their ease of handling [24]. Glycerin was selected as the co-surfactant due to its superior oil-incorporation capacity (158.67 ± 2.35 mg/g) in comparison to propylene glycol (90.37 ± 1.75 mg/g). Poloxamer 407 was excluded from further consideration due to its solid state and instability caused by gelation upon exceeding its sol-gel transition temperature (Tsol-gel) under laboratory conditions [25]. Consequently, coconut oil, polysorbate 80, and glycerin were selected as the oil, S/CoS, respectively, for the ME formulation.
3.2. Formulation of ME
The determination of the existence zones of the MEs was achieved through the execution of aqueous titrations, employing the selected components: coconut oil as the oil phase and a polysorbate 80/glycerin mixture as the S/CoS system. The mole fraction of each component was calculated based on the titration endpoint, which is defined as the point where the ME breaks or the phases separate. Using these data, pseudoternary diagrams were created (Fig. 1). In these diagrams, the shaded area indicates the existence zones of the ME. In contrast, the rest of the diagram corresponds to biphasic, turbid, or traditional emulsions, as determined through visual observation.
 | Figure 1. Pseudoternary phase diagrams of MEs, S/CoS ratio (1:1); (1:2); (1:3); (2:1); (3:1), A% (S/CoS); B% (Water); C% (Oil). [Click here to view] |
The ME zone exhibited an expansion that was proportional to the increase in the S/CoS ratio, reaching its maximum at a ratio of 3:1. This ratio was thus employed to delineate the ME optimization limits.
3.2.1. ME component optimization
The D-optimal mixture design was used to optimize the ME. This kind of experimental design is employed by its known advantages: it is a cost-effective design to study the effect of numerical as well as categorical variables with reduced experimental runs, have a high predictive accuracy and it allows to explore how a chosen response is controlled by modifications in the tested factors, levels, and predicted responses [26].
The component levels for the design were derived from the ME zones of the pseudoternary phase diagram (3:1), as illustrated in Figure 1. The primary goal was to incorporate the oil phase at the highest possible ratio (20%) to ensure complete solubilization of the extract within this phase. This factor decisively governed the subsequent selection of the remaining component proportions.
The optimization of the ME formulation of A. peruviana extract was achieved by employing the levels delineated in Figure 1 to generate the DOMD through thirteen distinct experiments (Table 3). The study evaluated three independent variables: coconut oil (X1), water (X2), and the S/CoS ratio (X3). The responses or dependent variables analyzed included the droplet size (Y1), extensibility (Y2), and physical stability, which was evaluated using heat shock (Y3) and centrifugation (Y4) tests. The coding system employed in this study utilized a value of one (1) to denote stable MEs, while a value of zero (0) indicated formulations with phase separation or turbidity. This coding system enabled clear classification between stable and unstable formulations.
Table 3. Experimental design for A. peruviana microemulsion.
| Experiment | Independent variable | Response variable | |||||
|---|---|---|---|---|---|---|---|
| X(1) (%b/w) | X(2) (%b/w) | X(3) (%b/w) | Y(1) (nm) | Y(2) (mm) | Y(3) | Y4 | |
| 1 | 9.17 | 14.17 | 76.67 | 155.49 | 1,288.38 | 1 | 1 |
| 2 | 20.00 | 2.00 | 78.00 | 153.96 | 1,429.82 | 0 | 0 |
| 3 | 5.58 | 11.08 | 83.33 | 156.31 | 1,278.24 | 1 | 1 |
| 4 | 8.58 | 8.08 | 83.33 | 145.25 | 1,310.24 | 1 | 1 |
| 5 | 20.00 | 13.00 | 67.00 | 157.73 | 847.25 | 1 | 1 |
| 6 | 14.58 | 13.58 | 71.83 | 140.68 | 1,047.53 | 1 | 1 |
| 7 | 5.58 | 22.08 | 72.33 | 131.17 | 821.66 | 1 | 1 |
| 8 | 6.08 | 22.08 | 71.83 | 150.19 | 699.20 | 1 | 1 |
| 9 | 8.00 | 2.00 | 90.00 | 159.12 | 1,267.96 | 0 | 0 |
| 10 | 14.58 | 8.08 | 77.33 | 150.19 | 1,246.23 | 1 | 1 |
| 11 | 2.00 | 30.00 | 68.00 | 150.19 | 630.55 | 1 | 1 |
| 12 | 2.00 | 8.00 | 90.00 | 140.68 | 1,114.35 | 1 | 1 |
| 13 | 3.00 | 30.00 | 67.00 | 150.19 | 714.78 | 1 | 1 |
Independent variables: X1: coconut oil, X2: water, X3: S/CoS. Dependent variables Y1: droplet size, Y2: extensibility, Y3 and Y4: physical stability.
3.2.2. Evaluation of response variables of DOMD
A mixture design approach was employed to investigate the influence of formulation composition on the physicochemical and stability properties of the developed ME system. Three formulation components were considered as independent variables: coconut oil (X1), water (X2), and Co/S, X3. Due to the nature of mixture systems the sum of the component proportions was constrained to 100%, and each component was evaluated within predefined compositional limits to ensure feasibility and ME formation.
The experimental region was defined by the following constraints: coconut oil (2%–20% w/w), water (2%–30% w/w), and Co/S (60%–90% w/w). A D-optimal mixture design was generated using Minitab® software to efficiently explore the constrained experimental domain while minimizing the number of experimental runs. Thirteen experimental formulations were prepared and characterized according to the design matrix.
Four response variables were evaluated: droplet size (Y1, nm), extensibility (Y2, mm), thermal stability (Y3), and centrifugation stability (Y4). Droplet size and extensibility were treated as continuous responses, while thermal and centrifugation stability were recorded as binary pass/fail outcomes (1 = stable; 0 = phase separation or turbidity observed).
Quadratic Scheffé mixture models were fitted for the continuous responses (Y1 and Y2), according to the general form:
Y= b1x1 + b2x2 + b3x3 + b12x1x2 + b13x1x3 + b23x2x3
where x1, x2, and x3 represent the proportions of oil, water, and Co/S, respectively, b1, b2, and b3 are the linear regression coefficients describing the individual effects, while b12, b13, and b23 represent the interaction effects between the formulation components.
3.2.2.1. Droplet size (Y1)
Pseudoternary phase diagrams were constructed using the water titration method to determine the concentration range of the components of ME A. peruviana seeds, demonstrated the interaction between the independent and dependent variables. In the case of Y1, critical factor in ME systems, as it affects their stability and oil-incorporation capacity; the results showed that a reduced water and oil phases led to a decrease in the droplet size, while the S/CoS mixture exhibited a more pronounced effect. This phenomenon occurs because the reduction of the aqueous and oily phases promotes the uniform dispersion of the oil into smaller droplets. These droplets are stabilized by the interface created by the surfactants. As demonstrated in previous studies, lower oil content has been shown to limit droplet coalescence, leading to the formation of smaller-sized globules due to a denser and more stable interface [14,27].
3.2.2.2. Extensibility (Y2)
Regarding the Y2, related to the ease with which the ME spreads over a surface, this exhibited an increase concomitant with the rise in the S/CoS ratio, subsequently followed by the increase in the oily phase. This behavior suggests that an increase in S/CoS concentrations leads to a reduction in surface tension, thereby enhancing the mobility and fluidity of the system, as demonstrated by experiments 4 and 10. Conversely, the aqueous phase exhibited minimal influence, which is consistent with the extensive properties being more dependent on the cohesion between the oily and S/CoS phases than on the amount of water in the system [28].
3.2.2.3. Physical stability of the system (Y3)
Finally, Y3, evaluated by phase separation, was mainly influenced by the proportion of water. When the aqueous content was exceptionally low (2%, as observed in experiments 2 and 9), the system exhibited phase separation. This observation suggests the presence of a minimum threshold of water, which is indispensable for ensuring the structural stability of the o/w type system. This finding aligns with the conclusions of previous studies, which indicated that the stability of MEs is contingent upon a balanced distribution of phases and the appropriate adjustment of interfacial tensions between components [24].
In the case of DOMD, the ME formulation was tuned to maximize the properties of interest, including minimum droplet size, high extensibility, and maximum stability. The formulation was achieved with 13% water, 20% oil, and 67% S/CoS. This composition ensures a proper balance between the phases to avoid separation, maintain a robust interface, and facilitate its application (Fig. 3). The high proportion of S/CoS strengthens the interfaces between the droplets, thereby ensuring the physical stability of the system. Concurrently, the oil phase contributes to the extensibility of the final product (Fig. 2).
 | Figure 2. Effect of formulation variables on droplet size, extensibility, and physical stability. [Click here to view] |
 | Figure 3. Topical microemulsion of the ethanolic extract of A. peruviana seeds, A% (S/CoS); B% (Water); C% (Oil). [Click here to view] |
The preparation of the ME involved three sequential steps to ensure a homogeneous and stable integration of the system. First, the oily phase was prepared by mixing 20 g of coconut oil with 0.98 g of ethanolic extract of A. peruviana, ensuring adequate solubilization of the extract in the lipophilic matrix. Subsequently, the oil phase was incorporated into 67 g of the S/CoS mixture in a 3:1 ratio (polysorbate 80: glycerin), a combination that was selected for its ability to reduce interfacial tension, stabilize the oil droplets, and extend the ME region in the pseudoternary diagram. Polysorbate 80, a nonionic surfactant, is known to facilitate efficient emulsification, while glycerin is recognized for its hygroscopic properties, which enhance water retention and extensibility. Subsequently, 13 g of distilled water was added to the oil phase and S/CoS mixture, with continuous stirring to ensure uniform distribution of the phases and prevent separation. This addition sequence follows fundamental principles of ME formulation, with the objective of maximizing dispersion and thermodynamic stability of the system [29]. The final ratio of components was defined based on the experimental design, achieving a balance between reduced droplet size, high extensibility, and physical stability [14,24]. This base formulation will serve for the incorporation of additional excipients (Table 4), thereby enabling the adjustment of properties such as viscosity, pH, and stability. This adaptability in the formulation will facilitate the optimization of the ME for a range of pharmaceutical and cosmetic applications.
Table 4. Formulation of microemulsion with A. peruviana.
| Excipients | Function | % |
|---|---|---|
| Coconut oil | Solvent | 19.91 |
| Polysorbate 80 | Surfactant | 50.25 |
| Glycerin | Co-surfactant | 16.75 |
| Tocopherol | Antioxidant | 0.20 |
| EDTA | Chelating agent | 0.05 |
| Phenoxyethanol | Preservative | 0.80 |
| A. peruviana extract | Active | 0.98 |
| Water | Solvent | 11.00 |
3.3. Pharmaceutics characterization
The results of the physical characterization of A. peruviana seeds ME are presented in Table 5.
Table 5. Pharmaceutics characterization of the microemulsion containing A. peruviana.
| Pharmaceutics properties | Result* |
|---|---|
| Viscosity (cP) | 1660 ± 25 |
| pH | 5.5 ± 0.03 |
| Refractive index | 1.45 ± 0.01 |
| Droplet size (nm) | 110.07 ± 50.5 |
| Conductivity (µS/cm) | 17.42 ± 0.40 |
| Physical stability (heat shock) | Stable |
| Physical stability (centrifugal) | Stable |
* Each value represents the mean ± SD of three independent measurements (n = 3).
In previous studies, the viscosity of ME systems has been reported to range from 40 to 343 cP [30–34]. In the obtained formulation, the increase in viscosity can be attributed to the low water content and high concentration of S/CoS. Although this may appear disadvantageous, it actually contributes to the stability of the system. The high viscosity decreases the diffusion constant, thereby reducing the collision frequency between droplets. This, in turn, minimizes the risk of phase separation [35].
The optimized microemulsion exhibited a pH of 5.5 ± 0.03, which is highly compatible with the physiological pH of the skin surface, typically ranging between 4.5 and 6.0. Maintaining an acidic pH is essential for preserving the integrity of the stratum corneum, sustaining the skin’s protective acid mantle, and reducing the risk of irritation or barrier disruption. The measured pH, therefore, indicates that the formulation is suitable for topical application and is unlikely to cause discomfort or adverse reactions, supporting its potential as a safe vehicle for delivering the A. peruviana extract [35].
The refractive index was evaluated, yielding a value that closely approximates 1.333 (the refractive index of water), which is consistent with the transparency and clarity exhibited by the ME. These findings are in alignment with those reported in other investigations of analogous systems [31,36,37].
The mean droplet size was 110.070 nm, which is within the typical range for ME-type systems (10–140 nm). This range is adequate to ensure the stability and functionality of the system [38,39]. While a higher surfactant concentration has been demonstrated to reduce droplet size by improving interfacial stability, the obtained size ensures a balance between active ingredient release, absorption, and in vivo stability [40].
Finally, the conductivity value, expressed in µS/cm, was low, which may be due to the limited amount of water present in the formulation. This finding is in agreement with that reported previously [34]. However, other authors have not found a direct correlation between aqueous content and conductivity [33]. These results suggest that the developed ME exhibits physical and structural characteristics suitable for potential applications in pharmaceutical formulations.
3.4. Evaluation of cell safety and anti-inflammatory activity
3.4.1. Cell viability
The evaluation of cell viability using the MTT assay showed that the topical 1% formulation of ethanolic extract of A. peruviana does not have a significant effect on the viability of RAW 264.7 macrophages (Fig. 4). In this study, the results of cell viability assay of ME obtained, indicates a high level of biocompatibility and suggests that it could be used in further in vivo studies, meeting the criterion of noncytotoxicity (viability >80% of the control). It also implies that the excipients in the formulation are biocompatible. Overall, these findings suggest that any reduction in inflammatory mediators is due to the extract’s pharmacological activity rather than a cytotoxic effect [41].
 | Figure 4. Effect of different concentrations A. peruviana microemulsion (15 to 60 μg/ml) on RAW 264.7 macrophage viability. Data corresponds to the mean ± SEM of two independent experiments (n = 12). ****p < 0.0001 statistically significant, were determined using one-way ANOVA and Dunnett’s test compared against the without extract formulation-control group. [Click here to view] |
3.4.2. Anti-inflammatory activity (production of NO•)
In addition, anti-inflammatory activity (production of NO•) indicates that the observed inhibitory activity is solely due to the extract (Fig. 5). The significant NO• inhibition indicates an effect dependent on the bioactive metabolites of the extract, such as phenolic compounds and possible sesquiterpene lactones. These have been previously described in the literature as negative modulators of iNOS expression and neutralizers of ROS. Previous studies have also shown that the biological activity of the extract remains after storage, supporting its functional stability [9]. Previous results obtained for this extract on RAW 264.7 macrophages show potent activity against important mediators of the inflammatory process, such as NO•, IL-1β, IL-6, and TNF-α, without affecting cell viability, suggesting a favorable efficacy and safety profile under biological conditions [10].This activity was maintained in the developed microemulsion, which also exhibits desirable properties for this type of formulation, including adequate spreadability, viscosity, and physical stability, supporting its potential development as a phytopreparation for topical use. On this basis, it is proposed to move forward with preclinical studies in in vivo models of skin inflammation to confirm its efficacy and dermal safety, as well as to develop skin permeation studies. In the near future, all results obtained with this extract and its formulation could support the design of an initial exploratory clinical trial to assess safety and tolerability in humans, followed by proof-of-concept studies in populations with inflammatory dermatological conditions.
 | Figure 5. Effect of different concentrations A. peruviana microemulsion (15 to 60 μg/ml) on NO• release in RAW 264.7 macrophages stimulated with LPS (1 μg/ml). Data corresponds to the mean ± SEM of two independent experiments (n = 12). 1400 W (10 μM) was used as a positive control. ****p < 0.0001 statistically significant, were determined using one-way ANOVA and Dunnett’s test compared against the LPS-control group. [Click here to view] |
4. CONCLUSION
In this study, an ME for topical use containing an anti-inflammatory extract obtained from the seeds of A. peruviana was designed and optimized. Using a rational approach based on preformulation studies and DOMD, compatible excipients were selected, and an optimal composition (20% coconut oil, 67% S/CoS, and 13% water) was determined. This formulation produced a stable ME with a droplet size of approximately 110 nm, suitable viscosity, and good extensibility. A pharmaceutical characterization was performed to evaluate the physical stability of the formulation under thermal and mechanical stress conditions. This analysis revealed that the formulation exhibited adequate conductivity and transparency. Furthermore, in vitro studies confirmed the biocompatibility of the ME and its capacity to inhibit NO• production, an important mediator of inflammation. These findings support using MEs as effective delivery systems for the topical application of bioactive plant extracts, emphasizing the potential of A. peruviana seeds as a source of safe and potent anti-inflammatory compounds.
5. ACKNOWLEDGMENTS
The authors would like to thank the Research Division of the Universidad de Cartagena (Project Code: 125 -2021) for all the financial support provided to conduct this study.
6. AUTHOR CONTRIBUTIONS
All authors made substantial contributions to conception and design, acquisition of data, or analysis and interpretation of data; took part in drafting the article or revising it critically for important intellectual content; agreed to submit to the current journal; gave final approval of the version to be published; and agree to be accountable for all aspects of the work. All the authors are eligible to be an author as per the International Committee of Medical Journal Editors (ICMJE) requirements/guidelines.
7. CONFLICTS OF INTEREST
The authors report no financial or any other conflicts of interest in this work.
8. ETHICAL APPROVALS
This study does not involve experiments on animals or human subjects.
9. DATA AVAILABILITY
All data generated and analyzed are included in this research article.
10. PUBLISHER’S NOTE
All claims expressed in this article are solely those of the authors and do not necessarily represent those of the publisher, the editors and the reviewers. This journal remains neutral with regard to jurisdictional claims in published institutional affiliation.
11. USE OF ARTIFICIAL INTELLIGENCE (AI)-ASSISTED TECHNOLOGY
The authors declare that they have not used artificial intelligence (AI)-tools for writing and editing of the manuscript, and no images were manipulated using AI.
REFERENCES
1. Zhong J, Shi G. Regulation of inflammation in chronic disease. Front Immunol. 2019;10(737):737. CrossRef
2. Manabe I. Chronic inflammation links cardiovascular, metabolic and renal diseases. Circulat J. 2011;75(12):2739–48. CrossRef
3. Heo SJ, Yoon WJ, Kim KN, Ahn GN, Kang SM, Kang DH, et al. Evaluation of anti-inflammatory effect of fucoxanthin isolated from brown algae in lipopolysaccharide-stimulated RAW 264.7 macrophages. Food Chem Toxicol. 2010;48(8–9):2045–51. CrossRef
4. World Health Organization. Noncommunicable diseases [Internet]. Geneva, CH: World Health Organization; 2025 [cited 2025 Oct 11]. Available from: https://www.who.int/news-room/factsheets/detail/noncommunicable-diseases
5. Seyedsadjadi N, Grant R. The potential benefit of monitoring oxidative stress and inflammation in the prevention of non-communicable diseases (NCDs). Antioxidants. 2020;10(1):15. CrossRef
6. Argoff CE. Recent developments in the treatment of osteoarthritis with NSAIDs. Curr Med Res Opin. 2011;27(7):1315–27. CrossRef
7. Chaachouay N, Zidane L. Plant-derived natural products: a source for drug discovery and development. Drugs Drug Candidates. 2024;3(1):184–207. CrossRef
8. Talhouk RS, Karam C, Fostok S, El-Jouni W, Barbour EK. Anti-inflammatory bioactivities in plant extracts. J Med Food. 2007;10(1):1–10. CrossRef
9. Palacio Y, Castro JP, Bassani VL, Franco LA, Bernal CA. Preformulation studies for the development of a microemulsion formulation from Ambrosia peruviana All., with anti-inflammatory effect. Braz J Pharm Sci. 2023;59(3):1–4. CrossRef
10. Castro JP, Franco LA, Diaz F. Anti-inflammatory screening of plant species from the colombian caribbean coast. J Appl Pharm Sci. 2021;11(4):106–17. doi: https://doi.org/10.7324/JAPS.2021.110413
11. Ohadi M, Shahravan A, Dehghannoudeh N, Eslaminejad T, Banat IM, Dehghannoudeh G. Potential use of microbial surfactant in microemulsion drug delivery system: a systematic review. Drug Design Develop Therapy. 2020;14:541–50. CrossRef
12. Laothaweerungsawat N, Neimkhum W, Anuchapreeda S, Sirithunyalug J, Chaiyana W. Transdermal delivery enhancement of carvacrol from Origanum vulgare L. essential oil by microemulsion. Int J Pharm. 2020;579:119052. CrossRef
13. Hu Q, Fu XL, Dong YY, Ma J, Hua J, Li JT, et al. D-optimal design and development of a koumine-loaded microemulsion for rheumatoid arthritis treatment: in vivo and in vitro evaluation. Int J Nanomed. 2023;18:2973–88. CrossRef
14. Chouhan P, Saini TR. D-optimal design and development of microemulsion based transungual drug delivery formulation of ciclopirox olamine for treatment of onychomycosis. Indian J Pharm Sci. 2016;78(4):498–511. CrossRef
15. Carneiro AF, Carneiro CN, De N Pires L, Teixeira LSG, Azcarate SM, De S Dias F. D-optimal mixture design for the optimization of extraction induced by emulsion breaking for multielemental determination in edible vegetable oils by microwave-induced plasma optical emission spectrometry. Talanta. 2020;219:121218. CrossRef
16. Rajpoot K, Tekade RK. Microemulsion as drug and gene delivery vehicle: an inside story. In: Tekade RK, editor. Drug delivery systems. Cambridge, MA: Academic Press; 2019. pp. 455–520. doi: https:// doi.org/10.1016/B978-0-12-814487-9.00010-7
17. Petrochenko PE, Pavurala N, Wu Y, Yee Wong S, Parhiz H, Chen K, et al. Analytical considerations for measuring the globule size distribution of cyclosporine ophthalmic emulsions. Int J Pharm. 2018;550(1–2):229–39. CrossRef
18. Green LC, Wagner DA, Glogowski J, Skipper PL, Wishnok JS, Tannenbaum SR. Analysis of nitrate, nitrite, and 15Nnitrate in biological fluids. Anal Biochem. 1982;126(1):131–8. CrossRef
19. Boateng L, Ansong R, Owusu W, Steiner-Asiedu M. Coconut oil and palm oil’s role in nutrition, health and national development: a review. Ghana Med J. 2016;50(3):189–96. CrossRef
20. Castro G, Apostol J. Formulation and evaluation of virgin coconut oil cream for atopic dermatitis prepared using green processing method. Acta Manilana. 2017;65:1–9. CrossRef
21. Gunarto C, Ju YH, Putro JN, Tran-Nguyen PL, Soetaredjo FE, Santoso SP, et al. Effect of a nonionic surfactant on the pseudoternary phase diagram and stability of microemulsion. J Chem Eng Data. 2020;65(8):4024–33. CrossRef
22. Mohamad Ja’afar S, Mohd. Khalid R, Othaman R, Wan Mokhtar WNA, Ramli S. Coconut oil based microemulsion formulations for hair care product application. Sains Malays. 2019;48(3):599–605. CrossRef
23. Bernal CA, Baena CM, Urrego JR, Ahumada V. Micro y nano encapsulación. Avances en las industrias farmacéutica, cosmética, biotecnológica y alimentaria. 1st ed. Cartagena, Colombia: Universidad Cartagena; 2025.
24. Vlaia L, Coneac G, Mu? AM, Olariu I, Vlaia V, Anghel DF, et al. Topical biocompatible fluconazole-loaded microemulsions based on essential oils and sucrose esters: formulation design based on pseudo-ternary phase diagrams and physicochemical characterization. Processes. 2021;9(144):1. CrossRef
25. Fakhari A, Corcoran M, Schwarz A. Thermogelling properties of purified poloxamer 407. Heliyon. 2017;3(8):1–26. CrossRef
26. Selim HMRM, Gomaa FAM, Alshahrani MY, Aboshanab KM. Response surface D-optimal design for optimizing fortimicins production by Micromonospora olivasterospora and new synergistic fortimicin-a-antibiotic combinations. Curr Microbiol. 2025;82(2):68. CrossRef
27. Barot BS, Parejiya PB, Patel HK, Gohel MC, Shelat PK. Microemulsion-based gel of terbinafine for the treatment of onychomycosis: optimization of formulation using D-optimal design. AAPS PharmSciTech. 2012;13(1):184–92. CrossRef
28. Mcclements DJ, Jafari SM. Improving emulsion formation, stability and performance using mixed emulsifiers: a review. Adv Colloid Interface Sci. 2018;251:55–79. CrossRef
29. Zhu T, Kang W, Yang H, Li Z, Zhou B, He Y, et al. Advances of microemulsion and its applications for improved oil recovery. Adv Colloid Interface Sci. 2022;299:102527. CrossRef
30. Acharya SP, Pundarikakshudu K, Panchal A, Lalwani A. Development of carbamazepine transnasal microemulsion for treatment of epilepsy. Drug Del Translational Res. 2013;3(3):252–9. CrossRef
31. Bhatia G, Zhou Y, Banga AK. Adapalene microemulsion for transfollicular drug delivery. J Pharm Sci. 2013;102(8):2622–31. CrossRef
32. Fouad SA, Basalious EB, El-Nabarawi MA, Tayel SA. Microemulsion and poloxamer microemulsion-based gel for sustained transdermal delivery of diclofenac epolamine using in-skin drug depot: in vitro/in vivo evaluation. Int J Pharm. 2013;453(2):569–78. CrossRef
33. Moghimipour E, Salimi A, Leis F. Preparation and evaluation of tretinoin microemulsion based on pseudo-ternary phase diagram. Adv Pharm Bull. 2012;2(2):141–7. CrossRef
34. Patel V, Kukadiya H, Mashru R, Surti N, Mandal S. Development of microemulsion for solubility enhancement of clopidogrel. Iran J Pharm Res. 2010;9(4):327–34.
35. Callender SP, Mathews JA, Kobernyk K, Wettig SD. Microemulsion utility in pharmaceuticals: implications for multi-drug delivery. Int J Pharm. 2017;526(1–2):425–42. CrossRef
36. Tiwari N, Chopra H, Sagar M, Sharma AK. A review on self emulsified drug delivery system: a promising approach for drug delivery of BCS Class II and IV Drugs. J Pharm Res. 2016;15(4):181–8. CrossRef
37. Shinde UA, Modani SH, Singh KH. Design and development of repaglinide microemulsion gel for transdermal delivery. AAPS PharmSciTech. 2018;19(1):315–25. CrossRef
38. Sharma P, Mittal A, Bajpai M. A study of oleic acid oily base for the topical delivery of dexamethasone microemulsion formulations. Asian J Pharm. 2009;3(3):208–14. CrossRef
39. Venkatesh G, Majid MIA, Mansor SM, Nair NK, Croft SL, Navaratnam V. In vitro and in vivo evaluation of self-microemulsifying drug delivery system of buparvaquone. Drug Dev Ind Pharm. 2010;36(6):735–45. CrossRef
40. Lee BS, Kang MJ, Choi WS, Choi YB, Kim HS, Lee SK, et al. Solubilized formulation of olmesartan medoxomil for enhancing oral bioavailability. Arch Pharm Res. 2009;32(11):1629–35. CrossRef
41. Bahrami M, Ghazavi A, Ganji A, Mosayebi G. Anti-Inflammatory activity of S. marianum and N. sativa extracts on macrophages. Rep Biochem Mol Biol. 2021;10(2):288–01. doi: https://doi.org/10.52547/rbmb.10.2.288